Bot. Mag. Tokyo 84: 123-136 (March 25, 1971) Electron Microscope Studies on the Morphogenesis of Plastids V. Concerning One-dimensional Metamorphosis of the Plastids in Cryptomeriac Leaves* by Susumu TOYAMA** and Kazuo FUNAZAKIS** Received November 25, 1970 Abstract To know about the mechanism of interconversion of plastids, electron micro- scopic observations were made on Cryptomeria leaves which aquire a reddish brown color in winter and recover their green color in coming spring to summer. In normal green leaves, two different kinds of plastids have been observed, viz, the chloroplasts having well organized grana structure in mesophyll cells and those completely lacking grana lamellae in bundle sheath cells. General feature of plastids in reddish brown leaves, may be summarized as follows : (1) The presence of red granules of rhodoxanthin (a carotenoid), and a well developed lamellar system involving grana- and intergrana-lamellae. (2) The plastoglobules, osmio- philic granules in plastids, increase in number and size as compared with the chloroplasts in normal green leaves. (3) Shrinkage which is one of the character- istic features of senescent plastids is not observed. (4) RNA content in plastid stroma is almost unchanged throughout an entire leaf stage ranging from normal green to subsequent regreening. Basic structure of the plastids in regreened leaves is quite similar to that in winter leaves, except for some increase of thylakoid membrane and decrease of osmiophilic granules. Accordingly, it is presumed that the plastids appearing in reddish brown leaves are not mere chro- moplasts but those having an incipient nature of the chloroplast, since real chromoplasts can not be converted into the chloroplasts. It seems that monotrope Plastiden-Metamorphose " is plausible in this case. Until now, two controversial opinions have been presented as regards the metamorphosis of the plastids. According to Schimperl', three kinds of plastids, chloroplast, chromoplast and leucoplast, are capable of interconversion from one to another. However, Frey-Wyssling et al.2' are of the opinion that the metamorphosis of plastids proceeds only in a linear fashion. Recently, Toyama and Ueda3' have shown that the reversion of chromoplast into active chloroplast does not occur in autumn leaves of Ginkgo biloba. On the other hand, Thomson et al4' have demonst- rated the reversion of chromoplast into chloroplast in pericarp cells of Valencia orange (Citrus sinensis L.), whose color is orange in winter and green in the next spring to summer. Similar color change may also be observed in Cryptomeria * This work was presented at the 33 rd Annual Meeting of the Botanical Society of Japan held at Kumamoto on Nov. 1-3, 1968. ** Department of Botany , Faculty of Science, Tokyo Kyoiku University, Otsuka, Tokyo, Japan (Present address: Department of Botany, University of California, Davis, California 95616) *** Biological Laboratory , Jissen Joshi-Gakuen, Shibuya, Tokyo, Japan. 124 TOYAMA, S. and FUNAZAKI, K. Vol. 84 leaves, which become reddish brown in winter and undergo regreening in spring. Two mechanisms may be conceivable in the analysis of regreening phenomena in these plants. The one is a reversion of the chromoplasts into the chloroplasts, and the other is the de novo formation of chloroplasts from the proplastids replacing Fig. 1 Scheme of reversible meta- the collapsing chromoplasts. Using Crypto- morphosis of the plastids proposed by meria leaves, the present experiment was Schimper (1885). carried out in order to clarify the process, of plastid metamorphosis in detail and to determine the alternative of both mechanisms mentioned above. Fig. 2. Scheme of monotrope Plastiden-Metamorphose " proposed by Frey-Wyssling et at. (1955). Materials and Methods Leaves of Cryptomeria japonica were collected at two different locations,, at Yunoyama in Mie Prefecture on March, 1965 and at Higashimatsuyama in Saitama Prefecture on February, 1968. Some of the fresh leaf samples were used immediately for electron microscopic observation, and the other leaves were cultured in tap water under continuous illumination (fluorescent light, 25001ux) at ca. 20°. In order to examine the membrane system within the plastids, small leaf pieces were fixedd with 1 solution of KMnO4 for 2 hours at 4° . For the observation of granules and fine structure in the stroma, samples were prefixed with 3.5°Q solution of glutaral- dehyde for 1 hour and post-fixed with 2°o aqueous 0504 for 2 hours at 4° . Thenn the fixed materials were dehydrated with acetone series, embedded in epoxy-resin - and polymerized at 50-60 ° . Sections were cut with an ultramicrotome (Porter-Blum, . Type MT-P, and examined with an electron microscope (Type JEM-7). Sections were stained withh a saturated solution of uranyl acetate for 2 hours and then with Millonig's solutions' for 30 minutes. Cytochemical examination on the senescence of plastids was made by the use of light microscope. For this purpose, several sections of fresh materials were stained with azure-B for the discrimination of RNA, and with fast green at pH 2 for the evaluation of total protein (Shaw et al.)6'. The sections to be stained with azure-B were treated in advance with DNA-ase to remove DNA. The staining with fast green was carreid out after removal of both DNA and RNA by pretreatment with 5% trichloroacetic acid. March, 1971 Electron Microscope Studies on the Morphogenesis of Plastids 125 Results 1. Plastids in normal green leaves In green leaves of Cryptomeria japonica, two different kinds of chloroplast were observed. The chloroplasts in mesophyll cells such as seen in photo 1, are typical ones with respect to their size and internal structure. These chloroplasts possess not only well-developed grana stacks connected with intergrana-lamellae but also plastoglobules and other fine granules. Another sort of chloroplast is shown in photo 2. The most remarkable feature in this chloroplast is a lack of grana structure. The internal lamellae are composed in general of a series of uninter- rupted membrane running in parallel along the long axis of the chloroplasts. In common to both kinds of chloroplast, starch grains, plastoglobules and ribosome like granules are contained, irrespective of the conspicuous difference in lamellar system. All leaves of Cryptomeria do not always attain a reddish brown color in winter. The leaves in the shade remain to be green as before. In these leaves, all plastids possess well-developed membrane structure together with a small number of plastoglobules (photo 3). 2. Plastids in reddish brown leaves Typical plastids appearing in the course of leaf color change, from green to reddish brown, are shown in photo 4. These plastids are characterized by gradual increase in number and size of plastoglobules and formation of small vacuoles in a periphery of stroma region. All plastids in reddish brown leaves possess more or less complicated lamellar system involving typical grana- and intergrana-lamellae, whose size is almost same as that in the normal green leaves (Table 1). Upon fixation with a solution of KMnO4, plastids usually retain their membrane system in an intact state (photo 5), but in some cases they are devoid of fine structure in the stroma. The most distinctive feature in the plastids of reddish brown leaves consists in an existence of numerous plastoglobules in the stroma. As a rule, 40- 80 globules of 0.1-0.5, t in size may be seen in one section of a plastid (photo 6 and 15). These globules are arranged among thylakoids often in a row along a longi- tudial axis of each plastid (photo 3, 15 and 18). Upon KMnO4 fixation spherical globules are disrupted into irregular, star-shaped bodies (photo 5, 7 and 16). In reddish brown leaves obtained from Yunoyama, most of the membrane systems in plastids have been disrupted into many discrete vesicles of ca. 0.05 i in Table 1. Some charcteristic features of the plastids in Cryptomeria leaves from summer to spring. 126 TOYAMA, S. and FUNAZAKI,K. Vol. 84 size. These minute bodies may be regarded as a constituent of the thylakoid (photo 11). The vesicles are enveloped with double membrane and seem to be for- med from thylakoids one after another by successive constriction of its terminal part. Accordingly, it may be that the collapse of thylakoid membrane is a coun- terpart of the process of lamellar formation (Toyama et al .)3. Osmiophilic globules are also observed in the matrix of cytoplasm. They are larger in size (1-2~€ in diameter), smaller in number than the plastoglobules, and found often in contact with the plastids (photo 13). Reddish brown leaves, which had been stored in an ice-box for 4 months after collection, were gradually brought into regreening upon culture in the light at 20 ° . Plastids in such leaves contain a large number of plastoglobules together with a few membrane system (photo 15). Abnormal lamellar systems were also observed in the plastids, which seems to be derived from the plastids in bundle sheath cells (photo 14). In some plastids, many vacuoles have been formed seemingly due to the swelling of the thylakoid membrane (photo 16). By the examinations under the light microscope, red granules of carotenoids (mainly rhodoxanthin)7' were observed, 6-12 pieces in a single plastid of reddish brown leaves (Table 1). However, electron micrographs have shown that they were lost during the course of dehydration with acetone. The presence of grana is dif- ficult to confirm under the light microscope because of the concurrence of carotenoid granules within the plastids, while it is discernible with ease under the electron microscope. Throughout the whole process from normal green to regreening of leaves, the changes in size and number of plastoglobules are shown in Fig. 3. 3. Plastids in the leaves undergone regreening Reddish brown leaves of Cryptomeria recover their capacity of greening during the culture for 4-5 days under 2500 lux at 20° .